The analysis of complex natural mixtures presents a significant scientific challenge in biomedical studies involving natural products and other complex biogenic samples, particularly when the need for isolation of minor constituents arises. Countercurrent chromatography (CCC) offers high loading capacity, complete sample recovery, and high purity fractions. It makes use of a gentle liquid stationary phase that prevents degradation of labile samples, and is already a valuable tool in natural products research. CCC separations consume relatively little solvent and don't require the use of expensive solid supports, making them highly economical, both environmentally and fiscally. The frugal use of consumables makes CCC much more "green" than other competing technologies. Determination of partition coefficients (K-values) is a unique analytical capability offered by CCC. However, accurate measurement of analyte partition coefficients with modern CCC instruments requires that sample loading be constrained far below system capacity. With current technology, the unique practical advantages of CCC are essentially lost when the conditions are optimized to determine K- values. In addition, successful use of CCC currently requires specialized training and considerable operator time. Automation, as it has for many other technologies, could effectively mitigate both the academic burden of assimilating decades of key information spread over hundreds of journal articles, and the practical burden of operating a complex multi-component system. Challenges imposed by the dynamic nature of the liquid CCC column had prevented the development of automated CCC until the SBC achieved a breakthrough proof of concept during Phase I of this SBIR project. The long-term objective of this project, and the ultimately marketable product, will be a fully functional automated CCC controller that will provide the separation power of CCC to minimally trained users, as well as enhance current capabilities and provide new functionality to experienced users. Automated CCC will provide a significant contribution to the goals of the National Institutes of Health (NIH), empowering biomedical researchers in their quest to analyze complex bioactive samples and alleviate human disease. The success of this project will be demonstrated by its application to the purification and study of bioactive natural products present in commonly consumed botanical products, one of which has shown promise in the fight against tuberculosis. PUBLIC HEALTH RELEVANCE: The analysis of highly complex chemical mixtures presents a significant scientific challenge, particularly in biomedical studies involving natural products and other complex biogenic samples. Countercurrent chromatography (CCC) is a technique, originally developed at NIH, which is able to solve many challenges in this area. This SBIR research proposal provides the key piece of technology required to bring the immense potential of CCC's analytical capabilities to realization, empowering biomedical researchers with a much-needed new tool in the arsenal to combat disease through biochemical discovery.